PPI-based screening of hub genes related to sepsis migration/pyroptosis and immune infiltration analysis

Xu Ma; Jianhao Wang; Kun Han; Jinshuai Lu

doi:10.1371/journal.pone.0336982

Peer Review History

Original SubmissionJune 30, 2025
29 Aug 2025 Decision Letter - Zhanzhan Li, Editor Dear Dr. Xu, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by Oct 13 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols . We look forward to receiving your revised manuscript. Kind regards, Zhanzhan Li Academic Editor PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf . 2. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. 3. Please note that PLOS One has specific guidelines on code sharing for submissions in which author-generated code underpins the findings in the manuscript. In these cases, we expect all author-generated code to be made available without restrictions upon publication of the work. Please review our guidelines at https://journals.plos.org/plosone/s/materials-and-software-sharing#loc-sharing-code and ensure that your code is shared in a way that follows best practice and facilitates reproducibility and reuse. 4. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match. When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section. 5. If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise. Additional Editor Comments: Reviewer #1: Reviewer #2: [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? Reviewer #1: Yes Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? -->?> Reviewer #1: Yes Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available??> The PLOS Data policy Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: Yes Reviewer #2: Yes ****** Reviewer #1: 1. Strengths The bioinformatics pipeline (e.g., batch correction with sva, DEG analysis with limma, enrichment with clusterProfiler) is standard and well-executed, with external validation adding credibility. Results are logically presented, and the identification of 8 validated hub genes (TLR2, SIRT1, PTGS2, MAPK14, IL18, ICAM1, CD274, CASP3) is supported by ROC curves (AUC >0.7). Data availability is fully met, and no funding/competing interests are declared. -Major Concerns and Suggestions Methods and Results Soundness: The methods are detailed and reproducible, but some choices (e.g., lenient DEG threshold of logFC >0) could introduce noise. Conclusions are supported, but interpretations occasionally overreach (e.g., miRNA's "critical role" without direct evidence). Discussion Section Suggestions: The Discussion is comprehensive, linking results to sepsis biology and acknowledging limitations (e.g., dataset heterogeneity, lack of wet-lab validation). However, it could be more concise (reduce repetition of intro material) and focused on interpreting results within the study's scope. Strengthen by: Explicitly tying hub genes to sepsis mechanisms without overgeneralizing (e.g., specify that findings are from blood-derived data, limiting tissue-specific inferences). Expanding on implications for bioinformatics in sepsis research, emphasizing how this integrates pyroptosis/migration (supported by your enrichments in IL-17/TNF pathways). Discussing whether the 8 hub genes are novel in the sepsis context or already known in multiple inflammatory settings (e.g., ICAM1 and CD274 are well-established in endothelial activation and immune checkpoint roles across infections and cancers, as per Meng et al., 2016, Genet Mol Res, and Masugi et al., 2017, Gut). Clarifying if the study suggests causal mechanisms (e.g., via pathway enrichments) or merely associations (e.g., correlations in immune infiltration), to avoid implying untested causality. Further Analyses to Enhance Robustness (Not Currently Performed): To bolster the analysis, consider these additions, which are common in bioinformatics studies and would improve confidence in results without requiring new data: Weighted Gene Co-expression Network Analysis (WGCNA): Identify co-expression modules among the 23 DEGs and correlate with sepsis traits (e.g., severity). This could reveal if hub genes cluster together, strengthening PPI findings (e.g., as in Li et al., 2023, Front Immunol on pyroptosis-related genes). Single-Cell RNA-Seq Integration: Map bulk DEGs to public scRNA-seq sepsis datasets (e.g., GSE167363) to resolve cell-type specificity (e.g., pyroptosis in neutrophils vs. macrophages). Survival Analysis: If metadata allows (e.g., from GSE57065), perform Kaplan-Meier on high/low hub gene expression for mortality prediction. Alternative Cutoff Testing: Perform and report DEG analysis with stricter logFC cutoffs (e.g., \|logFC\| >0.5 or >1), as low thresholds (\|logFC\|>0) can inflate gene lists and FDR. Validation Across Subgroups: Where data allows, repeat analyses on subsets (e.g., adult vs. pediatric, early vs. late sepsis, infection source) to assess robustness of hub gene selection across patient subtypes. Methodological Comparison: Conduct pathway enrichment with alternative tools (enrichR, g:Profiler) to confirm core findings are not tool-dependent. Network Analysis Robustness: Re-run hub selection using multiple network centrality metrics (Degree, Closeness, Betweenness) and show overlap with the presented results. Sensitivity analyses test result stability under varied parameters. Suggested ones: DEG Threshold Variation: Re-run with logFC >0.5 or >1 to assess if the 23 DEGs/hubs change (common to check robustness, e.g., in Gao et al., 2024, Gene). Bootstrap Resampling for Enrichments: Bootstrap GO/KEGG (1000 iterations) to compute confidence intervals for p-values, reducing false positives from small gene sets. PPI Interaction Score Variation: Test STRING scores >0.7 (high confidence) vs. 0.4 to evaluate hub stability. Cross-Validation in ssGSEA: Split data into training/test sets for immune correlations to avoid overestimation. Partial Rank Correlation or Variance-Based Sensitivity Analyses: Apply in gene/pathway prioritization steps to check for consistent ranking under parameter perturbations. How were quality control (QC) checks performed before and after normalization? Were outlier samples removed? Were PCA plots quantitatively assessed (variance explained by PC1/PC2 before vs. after correction) rather than visually? Was log2 transformation applied consistently? Which immune cell signature set was used for ssGSEA—LM22 (CIBERSORT) or another? Was it validated for whole blood transcriptomics? Were multiple testing corrections applied to the gene–cell correlation analyses? Could strong positive/negative correlations be due to overall neutrophil or lymphocyte proportions rather than functional associations? Reviewer #2: Comments to the Author I appreciate the opportunity to review this manuscript, which addresses a highly relevant clinical issue: the identification of key genes related to sepsis, cell migration, and pyroptosis through bioinformatics analysis. The study is well-structured and represents a potentially valuable contribution to the understanding of sepsis pathophysiology. Below I provide my observations. Strengths Clear and specific title, focused on the central aim of the study. The introduction adequately contextualizes the clinical relevance of sepsis and the need for new biomarkers. The importance of miRNAs, cell migration, and pyroptosis in the pathogenesis of sepsis is well justified. The bioinformatics methodology is robust, integrating multiple datasets and employing enrichment analyses, PPI networks, and external validation. Results are presented in a logical and progressive manner: Identification of 3,566 DEGs. Selection of 23 genes related to pyroptosis/migration. Validation of 8 key genes (TLR2, SIRT1, PTGS2, MAPK14, IL18, ICAM1, CD274, and CASP3). Relevant biological pathways (IL-17, TNF, TLR) are identified, and immune infiltration analysis highlights meaningful correlations. The conclusion appropriately proposes that the validated genes may represent potential biomarkers and therapeutic targets. Overall, the manuscript is clearly written, requiring only minor stylistic and grammatical adjustments for international publication. Areas for Improvement The main weakness is the lack of experimental validation (in vitro or in vivo), which limits the clinical translation of the findings. It is recommended to emphasize that results should be considered exploratory and require confirmation in experimental models. There is heterogeneity across datasets (different platforms and patient characteristics). Although batch correction was applied, a more detailed discussion of the potential impact of these differences would strengthen the manuscript. The discussion could be enhanced in two key aspects: Clinical applications, contrasting the proposed genes with currently used biomarkers (e.g., procalcitonin, CRP). The relationship with secondary complications of sepsis, such as cardiac dysfunction, renal injury, or lung injury. Including a perspective on epigenetic processes (particularly DNA methylation) would enrich the analysis, as these mechanisms regulate gene expression and may affect the identified genes. Relevant proteins involved in cell migration, such as matrix metalloproteinases (MMPs), were not considered. A brief discussion of their role would complement the analysis. Some network figures (PPI, KEGG, mRNA-miRNA) are complex and difficult to interpret; highlighting key genes more clearly would improve readability. The manuscript could benefit from reducing redundancies in the text (e.g., repeated gene lists in multiple sections) to improve conciseness. Overall Recommendation The manuscript presents a solid and well-structured bioinformatics analysis that identifies genes of interest in sepsis. However, to strengthen its scientific impact, I recommend: Expanding the clinical and comparative discussion. Emphasizing methodological limitations more explicitly. Refining text, figures, and references. With these improvements, the article could provide a significant contribution and serve as a solid foundation for future translational studies in sepsis ****** what does this mean? ). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy Reviewer #1: Yes: Ali Afkhaminia Reviewer #2: Yes: César Bermúdez-Mejía ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org . Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0336982.r001
Revision 1
10 Oct 2025 Author Response Dear Editor / Editor and Reviewers Manuscript ID: PONE-D-25-35332 Title: PPI-based screening of hub genes related to sepsis migration/pyroptosis and immune infiltration analysis We are deeply grateful to you and the other reviewers for your invaluable comments and suggestions, which have played a crucial role in significantly improving the quality of our manuscript. In response to the thorough and insightful feedback provided, we have conducted extensive revisions to the paper. We have carefully addressed each of the points raised, supplemented relevant content where necessary, and refined several sections to enhance clarity and completeness. A detailed point-by-point response to all the reviewers’ comments has been prepared, and all changes in the manuscript have been highlighted in red for your convenience. We sincerely hope that the revised version meets with your approval and are more than willing to make any further adjustments as needed. Thank you very much for your time and support—it is a great honor to have our work considered for publication in PLOS ONE. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at RE�We sincerely appreciate your thorough review and valuable comments. We will meticulously revise the manuscript in strict accordance with the latest PLOS ONE template, comprehensively reviewing and adjusting the manuscript structure, formatting, file naming, figure/table uploads, captions, and reference style to ensure all submitted materials fully comply with the journal's requirements. Thank you once again for your valuable suggestions. 2. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. RE�Thank you for your careful review and valuable comments. We will supplement the ORCID iD of corresponding authors in the author information section in subsequent revisions, and ensure that verification is completed in the Editorial Manager system to meet journal-related requirements. Thank you again for your valuable advice. 3. Please note that PLOS One has specific guidelines on code sharing for submissions in which author-generated code underpins the findings in the manuscript. In these cases, we expect all author-generated code to be made available without restrictions upon publication of the work. Please review our guidelines at https://journals.plos.org/plosone/s/materials-and-software-sharing#loc-sharing-code and ensure that your code is shared in a way that follows best practice and facilitates reproducibility and reuse. RE�Thank you for your thorough review and valuable comments. All analytical procedures in this study were performed using R scripts developed by the authors. The relevant code and documentation have been prepared in accordance with the journal’s requirements. Upon publication of the manuscript, all author-generated code and associated documentation will be made publicly available on a mainstream open-source platform. Detailed access information will be provided in the “Data and Code Availability Statement” section of the subsequent manuscript to ensure the reproducibility and reusability of the research findings. We sincerely appreciate your insightful suggestions. 4. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match. When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section. RE�Thank you for your thorough review and valuable feedback. We will carefully verify and standardize the funding information in the subsequent revision to ensure consistency between the "Funding Information" and "Financial Disclosure" sections. Once again, we appreciate your insightful suggestions. 5. If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise. RE�We sincerely appreciate your thorough review and valuable suggestions. Based on your recommendations, this study will conduct a comprehensive evaluation of the relevance of the suggested literature and will incorporate appropriate citations in subsequent revisions as needed, thereby ensuring the scientific rigor and representativeness of the references cited. Thank you once again for your insightful comments. Additional Editor Comments: Reviewer #1: Reviewer #2: [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: 1. Strengths The bioinformatics pipeline (e.g., batch correction with sva, DEG analysis with limma, enrichment with clusterProfiler) is standard and well-executed, with external validation adding credibility. Results are logically presented, and the identification of 8 validated hub genes (TLR2, SIRT1, PTGS2, MAPK14, IL18, ICAM1, CD274, CASP3) is supported by ROC curves (AUC >0.7). Data availability is fully met, and no funding/competing interests are declared. -Major Concerns and Suggestions Methods and Results Soundness: The methods are detailed and reproducible, but some choices (e.g., lenient DEG threshold of logFC >0) could introduce noise. Conclusions are supported, but interpretations occasionally overreach (e.g., miRNA's "critical role" without direct evidence). RE�We sincerely thank the reviewers for their careful reading and valuable comments. In the current analysis, a relatively lenient threshold of logFC > 0 was adopted, primarily due to the generally limited expression magnitude observed in sepsis blood samples. This threshold was used during the initial screening phase to ensure biological sensitivity. However, in subsequent analyses, multiple layers of filtering and validation using an independent external dataset were applied to enhance the reliability of the results and mitigate potential noise introduced by the initial screening. Regarding the role of miRNAs in sepsis, we will revise the manuscript accordingly to more cautiously emphasize the predicted significance of miRNAs as potential regulatory factors, while explicitly stating that further experimental validation is required to elucidate their specific mechanisms. Detailed modifications can be found in the Discussion section. Once again, we appreciate your insightful suggestions. Discussion Section Suggestions: The Discussion is comprehensive, linking results to sepsis biology and acknowledging limitations (e.g., dataset heterogeneity, lack of wet-lab validation). However, it could be more concise (reduce repetition of intro material) and focused on interpreting results within the study's scope. Strengthen by: RE�Thank you for your thorough review and valuable comments. We will revise the Discussion section in accordance with your suggestions by reducing redundant background information, placing greater emphasis on the core findings of this study and their scientific significance, and providing a more in-depth interpretation of the relevant signaling pathways and immune mechanisms. Specific revisions are detailed in the Discussion section. Once again, we sincerely appreciate your insightful recommendations. Explicitly tying hub genes to sepsis mechanisms without overgeneralizing (e.g., specify that findings are from blood-derived data, limiting tissue-specific inferences). RE�Thank you for your thorough review and valuable comments. All data analyses and conclusions presented in this study are based on human peripheral blood samples. The mechanistic inferences regarding the relevant hub genes are made solely in response to the molecular characteristics observed in the peripheral blood microenvironment. We will further emphasize the tissue origin of our findings—specifically, the peripheral blood—in key sections such as the abstract, discussion, and conclusion, to avoid extrapolation to other tissues or organs. Additionally, a corresponding limitation will be explicitly addressed in the discussion section. We once again express our gratitude for your insightful suggestions. Expanding on implications for bioinformatics in sepsis research, emphasizing how this integrates pyroptosis/migration (supported by your enrichments in IL-17/TNF pathways). RE�Thank you for your thorough review and valuable comments. In the revised manuscript, we will further incorporate bioinformatics approaches to explore in depth the role of pyroptosis and migration-related mechanisms in sepsis, as well as their regulatory significance within key inflammatory pathways such as IL-17 and TNF. Specific revisions are detailed in the Discussion section. Once again, we appreciate your insightful suggestions. Discussing whether the 8 hub genes are novel in the sepsis context or already known in multiple inflammatory settings (e.g., ICAM1 and CD274 are well-established in endothelial activation and immune checkpoint roles across infections and cancers, as per Meng et al., 2016, Genet Mol Res, and Masugi et al., 2017, Gut). RE�Thank you for your thorough review and valuable comments. In accordance with your suggestions, we have further clarified the existing research background of the eight hub genes in sepsis and other inflammatory conditions in the revised manuscript, supplemented relevant literature, and highlighted the positioning and novelty of this study. Specific revisions can be found in the Discussion section. Once again, we sincerely appreciate your insightful recommendations. Clarifying if the study suggests causal mechanisms (e.g., via pathway enrichments) or merely associations (e.g., correlations in immune infiltration), to avoid implying untested causality. RE�We sincerely thank the reviewers for their thorough evaluation and valuable comments. In both pathway enrichment analysis and immune infiltration analysis, this study was based on statistical correlations and did not make direct inferences regarding causal mechanisms. All results involving pathways and immune cells were described using terms such as “associated” or “enriched,” avoiding any implication of causality. In the Discussion and Conclusion sections, we further explicitly stated that the findings of this study are based on correlational analyses and do not infer causation, while also emphasizing the need for future validation through functional experiments. Relevant revisions have been made accordingly in the Discussion and Conclusion sections. Once again, we express our gratitude for the insightful suggestions. Further Analyses to Enhance Robustness (Not Currently Performed): To bolster the analysis, consider these additions, which are common in bioinformatics studies and would improve confidence in results without requiring new data: Weighted Gene Co-expression Network Analysis (WGCNA): Identify co-expression modules among the 23 DEGs and correlate with sepsis traits (e.g., severity). This could reveal if hub genes cluster together, strengthening PPI findings (e.g., as in Li et al., 2023, Front Immunol on pyroptosis-related genes). Single-Cell RNA-Seq Integration: Map bulk DEGs to public scRNA-seq sepsis datasets (e.g., GSE167363) to resolve cell-type specificity (e.g., pyroptosis in neutrophils vs. macrophages). Survival Analysis: If metadata allows (e.g., from GSE57065), perform Kaplan-Meier on high/low hub gene expression for mortality prediction. Alternative Cutoff Testing: Perform and report DEG analysis with stricter logFC cutoffs (e.g., \|logFC\| >0.5 or >1), as low thresholds (\|logFC\|>0) can inflate gene lists and FDR. Validation Across Subgroups: Where data allows, repeat analyses on subsets (e.g., adult vs. pediatric, early vs. late sepsis, infection source) to assess robustness of hub gene selection across patient subtypes. Methodological Comparison: Conduct pathway enrichment with alternative tools (enrichR, g: Profiler) to confirm core findings are not tool-dependent. Network Analysis Robustness: Re-run hub selection using multiple network centrality metrics (Degree, Closeness, Betweenness) and show overlap with the presented results. RE�Thank you for your thorough review and valuable feedback. In the current study, we have integrated multiple transcriptomic datasets for comprehensive analysis and validated key findings using external datasets, with a focus on the association between differentially expressed genes, protein–protein interaction networks, and immune infiltration. This approach demonstrates strong systematic and scientific rigor. Due to limitations in sample size, metadata completeness, and clinical information in the available datasets, relevant analyses have not yet been conducted. However, we fully acknowledge the importance of such analyses for in-depth mechanistic studies. In the future, we plan to incorporate more extensive clinical samples and multi-omics data to further expand related analyses, thereby continuously enhancing the breadth and depth of this research. Once again, we appreciate your insightful suggestions. Sensitivity analyses test result stability under varied parameters. Suggested ones: DEG Threshold Variation: Re-run with logFC >0.5 or >1 to assess if the 23 DEGs/hubs change (common to check robustness, e.g., in Gao et al., 2024, Gene). RE�Thank you for your thorough review and valuable feedback. In the analytical process of this study, a lenient threshold of logFC > 0 was adopted during the initial screening phase, primarily due to the objective characteristic of generally low expression levels of genes in sepsis blood samples. The rationale behind this setting was to maximize the retention of potential biological signals, ensure biological sensitivity in the preliminary screening res Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0336982.r002
4 Nov 2025 Decision Letter - Zhanzhan Li, Editor PPI-based screening of hub genes related to sepsis migration/pyroptosis and immune infiltration analysis PONE-D-25-35332R1 Dear Dr.Xu, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. For questions related to billing, please contact billing support . If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Zhanzhan Li Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author Reviewer #1: All comments have been addressed Reviewer #2: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions??> Reviewer #1: Yes Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? -->?> Reviewer #1: Yes Reviewer #2: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available??> The PLOS Data policy Reviewer #1: Yes Reviewer #2: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: Yes Reviewer #2: Yes ****** Reviewer #1: This is a strong revision. The authors have demonstrated a high level of engagement with the review process and have made meaningful improvements to their manuscript. The study presents a valuable bioinformatics resource that identifies a robust set of hub genes at the intersection of pyroptosis and cell migration in sepsis, with clear insights into their potential functional roles and regulatory networks. The responses to the reviewers are comprehensive and satisfactory. I believe the manuscript, with the revisions as outlined in the response letter, is now suitable for acceptance in PLOS ONE Reviewer #2: All observations have been fully addressed, resulting in a manuscript that is more solid in its technical and scientific aspects. ****** what does this mean? ). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy Reviewer #1: Yes: Ali Afkhaminia Reviewer #2: Yes: César Bermúdez-Mejía ******** https://doi.org/10.1371/journal.pone.0336982.r003
Formally Accepted
Acceptance Letter - Zhanzhan Li, Editor PONE-D-25-35332R1 PLOS ONE Dear Dr. Xu, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset You will receive further instructions from the production team, including instructions on how to review your proof when it is ready. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few days to review your paper and let you know the next and final steps. Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. You will receive an invoice from PLOS for your publication fee after your manuscript has reached the completed accept phase. If you receive an email requesting payment before acceptance or for any other service, this may be a phishing scheme. Learn how to identify phishing emails and protect your accounts at https://explore.plos.org/phishing. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Zhanzhan Li Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0336982.r004

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .